The invention relates to the involvement of spermidine synthase with the development of osteoarthritis and in cartilage rehabilitation. More particularly, the invention relates to methods of treatment, compositions and the use of spermidine synthase inhibitors in the treatment of osteoarthritis and cartilage damage associated therewith.
Osteoarthritis (OA) is a common, debilitating, costly, and currently incurable disease. Novel approaches to therapy are clearly required. The disease is characterized by abnormal functioning of chondrocytes, their terminal differentiation and initiation of osteogenesis within articular cartilage tissue, and breakdown of normal cartilage matrix. Genes whose products are involved in chondrogenesis and osteogenesis starting from the common progenitor cells, genes determining the terminal differentiation of chondrocytes and genes whose products trigger breakdown of the cartilaginous matrix are obvious candidates for therapeutic intervention.
Epidemiology of OA
OA, also erroneously called degenerative joint disease, represents failure of a diarthrodial (movable, synovial-lined) joint. In idiopathic (primary) OA, the most common form of the disease, no predisposing factor is apparent.
Secondary OA is pathologically indistinguishable from idiopathic OA but is attributable to an underlying cause. OA is the most common of all human joint disorders and is the most prevalent arthritic condition in the United States and around the world. Estimates of OA prevalence based on clinical evaluation in various studies show that more than 90% of the population over the age of 70 has OA. The invention is aimed at novel avenues of therapy and prevention of the disease.
Pathogenesis of OA
OA is a heterogeneous group of conditions that lead to joint symptoms and signs associated with defective integrity of articular cartilage, in addition to related changes in the underlying bone at the joint margins. OA may be either idiopathic (i.e., primary) or secondary to other medical conditions (inflammatory, biochemical, endocrine-related, metabolic, and anatomic or developmental abnormalities). Age is the most powerful risk factor for OA but major trauma and repetitive joint use are also important risk factors for OA. The pattern of joint involvement in OA is also influenced by prior vocational or avocational overload.
The disease has two general stages: (1) compensated and (2) decompensated. Currently, most investigators feel that the primary changes occur in cartilage extracellular matrix due to exogenous reasons (i.e., load, injury etc.). Then, a defect in the collagen network of the cartilage is apparent, and lysosomal enzymes and secreted proteases (MMPs, plasmin, cathepsins) probably account for the observed initial alterations in cartilage matrix. Their synthesis and secretion are stimulated by IL-1 or by other factors (e.g., mechanical stimuli). In the initial stage of disease, compensatory cellular response is activated. Secreted by chondrocytes, protease inhibitors like TIMP and PAI-1 work to stabilize the system by opposing the protease activity. Growth factors such as IGF-1 and TGF-xcex2 are implicated in repair processes that may heal the lesion or, at least, stabilize the process by activating proliferation of cells of chondrogenic lineage. Finally, this leads to the accumulation of hypertrophic chondrocytes. The latter cells have marked biosynthetic activity that is expressed in increasing the proteoglycan (PG) concentration, associated with thickening of the cartilage (xe2x80x9ccompensatedxe2x80x9d OA). The compensatory mechanisms may maintain the joint in a reasonably functional state for years. However, the repair tissue does not hold up and the rate of PG synthesis falls off with full-thickness loss of cartilage. This marks the decompensated stage of OA. Following the destruction of the articular cartilage, there is migration of progenitor cells to the sites of tissue damage. These cells proliferate and differentiate into four cell types: osteoblasts, chondroblasts, chondroclasts and fibroblasts, which combine to form bony structures called osteophytes which protrude into the joint space, thus inhibiting its movement. Finally, gradual replacement of cartilage with bone occurs.
The reason for this phenomenon is unknown. One possibility is that in OA, the normal inhibitory growth control of articular chondrocytes or synovial membrane fibroblasts is altered. This enables accumulation of two types of cells that cannot be found in normal articular cartilage: (1) immature mesenchymal and bone marrow cells with modified properties, and (2) hypertrophic articular chondrocytes. Previous results have clearly shown that hypertrophic chondrocytes may trigger osteogenesis by secretion of angiogenic and osteogenic factors. (Homer, A., Bishop, N. J., Bord S., Beeton, C., Kelsall, A. W., Coleman, N. and Compston, J. E. (1999). Immunolocalisation of vascular endothelial growth factor (VEGF) in human neonatal growth plate cartilage. J. Anat. 194: 519-524).
In OA, therapeutic interference may target three main processes:
inhibition of initial cartilage damagexe2x80x94one of the accepted therapeutic strategies, combining recommendations to reduce the physical pressure on the joint and treatment with inhibitors of metalloproteinases;
inhibition or attenuation of total cartilage destruction at later stagesxe2x80x94implies the therapeutic activation of processes connected to cartilage rehabilitation, namely, the promotion of proper differentiation of mesenchymal progenitors into mature chondrocytes capable of producing fully functional articular cartilage tissue;
inhibition or attenuation of osteophyte formation at the end stage of the diseasexe2x80x94implies the therapeutic inhibition of ectopic osteogenesis at the site of articular cartilage.
Therefore, the inventors set out to identify target genes that code for specific factors that stimulate or inhibit the differentiation of progenitor cells to chondrocytes and/or stimulate or inhibit the differentiation of progenitor cells to osteoblasts.
Changes in gene expression caused by IL-1, FGF-2 and mechanical stress, which are known osteogenic factors, may be connected to OA development and, therefore, should be opposed by therapeutic intervention. Surprisingly, it has been found by the present inventors that one of the genes that were upregulated by FGF-2 is the spermidine synthase gene. This implied that the spermidine synthase gene might be involved in the OA pathway.
Spermidine Synthase
Spermidine is one of three bioactive polyamines, the other two being putrescine and spermine. Polyamines constitute a group of cell components that are important in the regulation of cell proliferation and cell differentiation. Although their exact functions have not yet been clarified, it is assumed that polyamines play an important role in a number of cellular processes such as replication, transcription, and translation.
The polyamine biosynthetic pathway consists of two highly regulated enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase, and two constitutively expressed enzymes, spermidine synthase and spermine synthase. Spermidine synthase is a 74 kDa protein that catalyses the 3-aminopropylation of putrescine (1,4-diaminobutane) to produce spermidine. The biosynthesis of spermidine involves decarboxylation of S-adenosylmethionine (SAM) to S-adenosyl-3-methylthiopropanamine (decarboxylated SAM) by SAM decarboxylase, and decarboxylation of ornithine to putrescine by ornithine decarboxylase. Decarboxylated SAM then reacts with spermidine synthase to generate an aminopropylated form of the enzyme, which then transfers the aminopropyl group to putrescine to produce spermidine and 5xe2x80x2-methylthioadenosine (MTA). The active enzyme is a dimer of two identical subunits, requires no cofactors, and uses dcAdoMet as an aminopropyl donor and putrescine as the acceptor.
Putrescine, spermidine and spermine have been found in many living tissues, including cartilage. Their formation, catalyzed by ODC, has been observed during the induction of cartilage transformation in bone. Parathyroid hormone, which stimulates the synthesis of glycosaminoglycans, induces ODC activity and increases polyamine levels in differentiated rabbit costal chondrocytes in culture. Resting cartilage is devoid of putrescine. Ossifying cartilage contains more polyamines than the resting zone (based on tissue weight and DNA content). The amount of spermidine in the ossifying zone is 5-fold higher and that of spermine about 2-fold. The spermidine/spermine ratio is 1.7 in the ossifying cartilage and 0.69 in the resting zone. Only spermidine showed the capacity of displacing proteoglycan subunits from a column of Sepharose 4B-type II collagen (Franco Vittur et al. (1986). A possible role for polyamines in cartilage in the mechanism of calcification. Biochimica et Biophysica Acta 881:38-45).
The effect of polyamines on the interaction of proteoglycan units with collagen was studied by following the elution of proteoglycans from a column of Sepharose 4B-collagen loaded with proteoglycan subunits. While putrescine and spermine were without effect, spermidine showed a strong capacity in displacing proteoglycan subunits: about 90% of the proteoglycan subunits were removed from the column. Spermine and spermidine increased the activity of alkaline phosphatase produced from cartilage. Spermidine was observed in the cells of the resting zone of preosseous cartilage. Cell staining disappeared, approaching the zone of proliferating and columnar cells. Staining for spermidine is markedly evident in the matrix only at the limit of columnar cells where hypertrophy of chondrocytes initiates. Among the three polyamines, spermidine is the most abundant: a high molar ratio of spermidine/spermine has been taken as an index of rapid growth. The highest amount of spermidine is in the ossifying region.
Polyamine Metabolism
The synthesis of the precursors, putrescine and decarboxylated S-adenosylmethionine (dcAdoMet) is brought about by the action of two decarboxylases ornithine decarboxylase (ODC, EC 4.1.1.17) and S-adenosylmethionine decarboxylase (AdoMetDC, SamDC, EC 4.1.1.51). These enzymes are very highly regulated by means of both growth factors and other stimuli that increase their levels, and by polyamines themselves, which reduce their activity. The combined effect of these agents is to adjust the polyamine levels to that needed for cell growth and development. Alternation in the activities of ODC and AdoMetDC are the major forces in controlling polyamine levels. The activities of the aminopropyltransferases, putrescine aminopropyltransferase (PAPT, spermidine synthase, EC 2.5.1.6) and spermidine aminopropyltransferase (SAPT, spermine synthase, EC2.5.1.22) are controlled primarily through the availability of their substrates. In addition to their de novo synthesis within the cells, polyamines can also be obtained as a result of uptake by a specific transport system. This transport system is regulated both negatively by the intracellular polyamine content and positively by growth factors and oncogenes. The presence of the transport system and its enhanced activity as a result of polyamine depletion is a significant factor in ameliorating the effect of the inhibition of polyamine synthesis. The uptake of exogenous polyamines may be a critical factor in the lack of success in clinical trials of these inhibitors as anti-tumor agents. Finally, polyamine levels can be altered as a result of interconversion, oxidation and efflux. The oxidation of polyamines at the terminal nitrogen atoms is accomplished by Cu+2-containing oxidases that appear to be located primarily extracellularly, although their complete absence from the cell has not been established. Interconversion and efflux of polyamines from the cell is facilitated by means of the action of spermidine/spermine-N-acetyltransferase (SSAT, EC 2.3.1.57) which acetylates the aminopropyl end of the polyamines forming N-acetylspermine and N-acetylspermidine. These acetyl derivatives bind less tightly to cellular polyanions and are either excreted or rapidly metabolized. They are oxidized at the internal nitrogen atom by a FAD-dependent oxidase called polyamine oxidase (PAO) splitting of N-acetylaminopropanal and converting spermine Into spermidine and spermidine into putrescine. The limiting factor in this pathway is the activity of SSAT, which is normally very low but is induced greatly by an increase in the cellular content of polyamines or by the application of toxic stimuli, which lead to the release of polyamines from membranes and cellular organelles. Under physiological conditions, PAO has little or no activity against non-acetylated polyamines.
It is therefore an object of the invention to identify different spermidine synthase inhibitors and to evaluate their effect as potential drugs for inhibiting or delaying OA.
It is a further object of this invention to provide therapeutic agents for the treatment of OA and for use in cartilage rehabilitation.
These and other objects of the invention will be elaborated on as the description proceeds.
In a first aspect, the present invention relates to a method for the treatment of a subject in need of treatment for OA, this method comprising administering to said subject an amount of an inhibitor of spermidine biosynthesis sufficient to effect a substantial inhibition of spermidine biosynthesis so as to thereby treat the subject.
In a preferred embodiment the inhibitor administered to said subject is a spermidine synthase inhibitor.
According to a specifically preferred embodiment, the spermidine synthase inhibitor is an inhibitor that leads to inhibition of any one of chondrocyte proliferation, chondrocyte final differentiation, angiogenesis and osteoclastogenesis.
In yet another preferred embodiment the spermidine synthase inhibitor administered to said subject may be selected from the group consisting of adenosyl spermidine, AdoDATO, DCHA, trans-4-methylcyclohexylamine (4MCHA), cyclohexylamine, methylglyoxal bis-(cyclopentylamidinohydrazone) (MGBP), 2-mercaptopropylamine, N-chlorosulfonyldicyclohexylamine, 5xe2x80x2-((3-aminopropyl)ammo)-5xe2x80x2-deoxyadenosine, 1-aminooxyl-3-aminopropane, 5xe2x80x2-(isobutylthio) adenosine, 5xe2x80x2-(methylthio) adenosine and any functional homologs and analogs thereof.
The invention further relates to the use of an inhibitor of one or more steps in the polyamine biosynthetic pathway in the treatment of OA in a mammalian subject. This inhibitor, according to a preferred embodiment, may be an inhibitor of spermidine biosynthesis, and in a more preferred embodiment it may be a spermidine synthase inhibitor and should inhibit the accumulation of spermidine.
According to a specifically preferred embodiment, the spermidine synthase inhibitor used may be an inhibitor that leads to inhibition of any one of chondrocyte proliferation, chondrocyte final differentiation, angiogenesis and osteoclastogenesis.
The spermidine inhibitor used may be selected from known spermidine synthase inhibitors, such as those described hereinabove.
The present invention further provides an inhibitor of the polyamine biosynthetic pathway for use in the treatment of OA in a mammalian subject. This inhibitor may be, according to preferred embodiment, a spermidine synthase inhibitor. More particularly, the inhibitor to be used in the treatment of OA is a spermidine synthase inhibitor that leads to inhibition of chondrocyte proliferation, chondrocyte final differentiation, angiogenesis and osteoclastogenesis.
In yet another aspect, the present invention relates to the use of an inhibitor of the polyamine biosynthetic pathway in the preparation of a pharmaceutical composition for the treatment of OA in a mammalian subject. Preferably, the inhibitor used may be an inhibitor of spermidine biosynthesis, more preferably it may be a spermidine synthase inhibitor; most preferably, this inhibitor is an inhibitor that inhibits the accumulation of spermidine and that leads to inhibition of any one of chondrocyte proliferation, chondrocyte final differentiation, angiogenesis and osteoclastogenesis.
The inhibitor used in the preparation of a pharmaceutical composition for the treatment of OA may be, according to the present invention, selected from the spermidine synthase inhibitors disclosed above.
The present invention further provides a therapeutic composition for the treatment of OA. This composition comprises as an active ingredient an inhibitor of one or more steps in the polyamine biosynthetic pathway. More preferably, the inhibitor is an inhibitor of spermidine biosynthesis. Most preferably, the inhibitor is a spermidine synthase inhibitor.
According to a preferred embodiment, the composition of the invention may optionally further comprise pharmaceutically or veterinarily acceptable carrier, excipient and/or diluent.
Another aspect of the present invention relates to a method of preparing a therapeutic composition for the treatment of OA. This method of preparation comprises the steps of (a) identifying an inhibitor of a spermidine synthase that leads to inhibition of any one of chondrocyte proliferation, chondrocyte final differentiation, angiogenesis and osteoclastogenesis; and (b) admixing said inhibitor with a pharmaceutically acceptable carrier, excipient and/or diluent.
In one preferred embodiment of the present aspect, identification of a spermidine synthase inhibitor suitable for the preparation of a therapeutic composition for the treatment of OA, is performed by the steps of:
(a) obtaining a candidate spermidine synthase inhibitor; and
(b) evaluating the effect of said candidate inhibitor on any one of chondrocyte proliferation, chondrocyte final differentiation, angiogenesis and osteoclastogenesis by an evaluating method.
The evaluating method comprises the steps of:
i. providing a test system comprising DNA encoding spermidine synthase;
ii. contacting said system with the said test candidate spermidine synthase inhibitor under conditions which normally lead to expression of spermidine; and
iii. determining the effect of the test candidate inhibitor on an end-point indication as compared to a control, wherein said effect is indicative of inhibition of any one of chondrocyte proliferation, chondrocyte final differentiation, angiogenesis and osteoclastogenesis by the test candidate inhibitor.
In a specific embodiment, the test system used by the evaluating method according to the invention may be any one of in vitro cell culture, ex vivo cell culture, ex vivo organ culture and in vivo animal model. In yet another specific embodiment, the spermidine synthase expressed by said test system used in the method of the invention, may be expressed either endogenously or exogenously. This test system may optionally further comprise endogenous and/or exogenous agents that provide suitable conditions for the expression of spermidine and for the detection of an end-point indication for determining any one of chondrocyte proliferation, chondrocyte final differentiation, angiogenesis and osteoclastogenesis.
Depending on the test assay system chosen, inhibition of chondrocyte proliferation, chondrocyte final differentiation, angiogenesis and osteoclastogenesis can be observed in a variety of ways, including intracellular staining assays (including immunohistochemical) and assays affecting an observable parameter; e.g., a physiological readout, such as change in cell cycle.
According to a preferred embodiment, the test system used by the method of the invention for evaluating the effect of said candidate inhibitor is an in vitro transfected cell culture. The cells employed carry an exogenously expressed spermidine synthase.
In an alternative embodiment, the test system used by the method of the invention for evaluation purposes is an ex vivo bone culture, comprising endogenously expressed spermidine synthase. Preferably, the bone culture used is an embryonic bone culture.
Another alternative test system may be an in vivo system, which is an animal model system. According to the method of the invention, use of an animal model for evaluation purposes enables utilizing the development of arthritis as an end-point indication. Where used as an end-point indication, development of arthritis may be determined, for example, by measuring paw thickness of said animal. Any increase in the size of the paw that is less than the increase observed in a control is indicative of inhibition of chondrocyte proliferation, chondrocyte final differentiation, angiogenesis and osteoclastogenesis, or development of arthritis by the test candidate inhibitor.
In one preferred test system, an appropriate animal model may be a transgenic mouse.
In yet another preferred in vivo test system, an arthritic mammalian model expressing endogenous spermidine synthase may be used by the evaluating method of the invention. According to this embodiment, the arthritic animal enables utilizing the development of arthritis as an end-point indication.
According to a particularly preferred embodiment, the arthritic mammal may be an arthritic rat or an arthritic mouse.
A specifically preferred embodiment of the invention relates to method of preparing a therapeutic composition for the treatment of OA. This method comprises the steps of (a) identifying an inhibitor of a spermidine synthase that leads to inhibition of any one of chondrocyte proliferation, chondrocyte final differentiation, angiogenesis and osteoclastogenesis; and (b) admixing said inhibitor with a pharmaceutically acceptable carrier. Identification of a suitable inhibitor involves obtaining a candidate inhibitor and evaluating the effect of the specific candidate. According to this embodiment, a candidate spermidine synthase inhibitor may be obtained for further evaluation, by selecting an inhibitor from the group consisting of adenosyl spermidine, AdoDATO, DCHA, trans-4-methylcyclohexylamine (4MCHA), cyclohexylamine, methylglyoxal bis (cyclopentylamidinohydrazone) (MGBP), 2-mercaptopropylamine, N-chlorosulfonyldicyclohexylamine, 5xe2x80x2-((3-aminopropyl)ammo)-5xe2x80x2-deoxyadenosine, 1-aminooxyl-3-aminopropane, 5xe2x80x2-(isobutylthio) adenosine, 5xe2x80x2-(methylthio) adenosine and any functional homologs and analogs thereof.
Alternatively, a candidate inhibitor may be obtained for further evaluation, by a screening method for a substance that is an inhibitor of spermidine synthase. According to the invention, such screening method comprises the steps of:
(a) providing a mixture comprising spermidine synthase;
(b) contacting said mixture with a test substance under conditions which normally lead to biosynthesis of spermidine; and
(c) determining the effect of the test substance on an end-point indication, whereby inhibition of said end point is indicative of inhibition of spermidine synthase by the test substance. According to a specific embodiment of this alternative, the end point indication may be the presence of a product of the spermidine synthase catalytic reaction.
All the above and other characteristics and advantages of the invention will be further understood through the following illustrative and non-limiting description of preferred embodiments thereof, with reference to the appended drawings.